Endonuclease-barcoding

Abstract

Provided herein are methods and kits for labeling endonuclease-treated cells. The methods comprise: contacting the cells to be labelled with at least one endonuclease suitable for targeting a genomic region of interest, and first and second nucleic acids suitable for introducing one or more silent (or optionally non-silent) mutation(s) in the genomic region by homology-directed repair (HDR). The mutation(s) introduced by the first nucleic acid differ from the mutation(s) introduced by the second nucleic acid.

Description

SEQUENCE LISTING[0001]This application includes as the Sequence Listing the complete contents of the accompanying text file “SequenceListing_ST25.txt”, created Apr. 17, 2018, containing 17050 bytes, hereby incorporated by reference.FIELD OF THE INVENTION[0002]The invention relates to methods, compositions and kits for labelling and detecting endonuclease-treated cells, and most preferably eukaryotic cells.BACKGROUND OF THE INVENTION[0003]In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis.[0004]As an alternative to more conventional approaches, such as cDNA overexpre...

AI Technical Summary

Benefits of technology

The invention relates to methods for labeling and detecting cells that have been treated with endonuclease. The methods involve introducing silent mutations in a genomic region of interest using a combination of endonuclease and a nucleic acid. The invention also includes a kit for labeling and detecting cells treated with CRISPR / Cas system. The technical effects of the invention include improved accuracy and sensitivity in identifying cells with targeted genomic modifications.

Problems solved by technology

DNA repair through NHEJ frequently generates insertions or deletions (indels), which can alter the frameshift of a coding sequence, thus resulting in gene inactivation.

Yet, those DNA editing tools, which are based on protein-DNA recognition, tend to suffer (to varying degrees) from multiple drawbacks:

(i) they may lack flexibility and / or are not easy-to-use;

(iv) they may not be compatible with modifications that have a negative impact on cell growth.

One of the consequences of such drawbacks is the requirement to further select individual clones in which the HDR event has occurred.

However, despite the undeniable potential of this new technology, the typical strategies based on CRISPR / Cas9 still bear at least some of the above-mentioned drawbacks:first, this system can also tolerate a few mismatches between the sgRNA and its target sequence, which can still result in off-target DNA cleavage.

While tools have been designed to identify in silico such potential off-target sequences, occasional minor shifts in the secondary structure of the RNA-DNA duplex have been recently reported that could make such predictions more difficult;secondly, the efficiency of CRISPR / Cas9-mediated DNA editing can vary substantially, depending on the cell model, the sgRNA and the targeted DNA, so that in a given experiment only a fraction of cells within a population will contain the desired genetic modification;besides the potential issues related to clonal variability, this approach still requires a reasonably high efficiency of CRISPR-mediated editing, and it is obviously still not compatible with modifications that have a negative impact on cell growth.

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